Ifn γ pe cf594

PBMCs were cultured in RPMI-1640 medium (GIBCO) containing 10% fetal bovine serum and stimulated with anti-CD3/CD28 (2 and 5 μg/ml) antibodies or phorbol 12-myristate 13-acetate (PMA)/ionomycin (50 ng/ml and 1 μg/ml, respectively), plus Golgiplug (BD Pharmingen, San Diego, CA, USA) for 5 h. The cells were then surface stained with CD4-FITC, CD8-APC-H7, PD-1-PE and TIM-3-PE-Cy7, and intracellularly stained with IFN-γ-PE-CF594, TNF-α-PerCp-Cy5.5 or IL-2-PerCp-Cy5.5 (BD Pharmingen) antibodies. A violet amine reactive dye (Invitrogen, Grand Island, NY, USA) was used to assess cell viability.

Lung tissue was chopped and digested with collagenase D (1 mg/ml, Roche) and DNase I (10 μg/ml, Sigma-Aldrich) for 1 h at 37 °C with agitation. Next, lungs or spleens were passed through a 40-μm cell strainer to obtain a single-cell suspension, followed by RBC lysis. The cells were incubated with CD16/CD32 FcγRIII (1:100) to block IgG Fc receptors. Cells were incubated with LIVE/DEAD Aqua (Invitrogen), followed by surface staining with fluorochrome-conjugated anti-mouse Abs for various markers. To detect cytokines, cells were stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of brefeldin A (5 μg/ml) for 4 h at 37 °C. For detection of intracellular cytokines, cells were fixed in 2% PFA and permeabilized with 0.5% saponin (Sigma-Aldrich, Ireland), followed by staining with IL-17A–V450 and IFN-γ–PE–CF594 (BD Biosciences). To discriminate blood-borne circulating cells from lung-localized cells, we used a well-described approach in which anti-mouse PE-CD45 Ab (eBioscience) was administered i.v. to mice 10 min before they were euthanized and lungs were harvested^{74 (link)}. Circulating leukocytes, which are exposed to the antibody and are labeled, become CD45iv⁺, whereas tissue-infiltrated cells are “protected” from labeling and remain CD45iv⁻. Gating strategy is provided in the Supplementary Information, as well as Source Data files.

PBMCs were stimulated with anti-CD3/CD28 (2 and 5 μg/mL), plus Golgiplug (BD Pharmingen) for 5 h before intracellular staining Blimp-1-PE, IFN-γ-PE-CF594, and IL-2-PerCp-Cy5.5 (BD Pharmingen). For perforin study, perforin-PE-CF594 (BD Pharmingen) was used. A Fixable Viability Dye eFluor 450 (eBioscience) was used to assess cell viability.